The present invention relates to hybrid cells and methods of making and using hybrid cells. Perhaps the most substantial practical application of hybrid cells is the production of hybridomas, which are used to produce monoclonal antibodies. In addition, they are used in a variety instances for research purposes, but their broader application, for example, in a clinical treatment setting has heretofore not been practical. These clinical applications include the cellular vaccines for treating or preventing cancer and other disorders, as well as preventing transplant rejection. The present invention responds to such deficiencies by providing methods and reagents that make the broad applicability of hybrid cells a reality.
Recent advances in molecular immunology now make immunotherapy a truly viable option for the treatment of patients with cancer and metastatic disease. The past decade has seen the approval and introduction of several immunotherapeutic strategies for wide ranging use against several metastatic cancers, Parkinson et al., in CANCER MEDICINE, 4th ed., pp. 1213-1226 (Holland et al., eds. 1997). Perhaps the best known strategies include IL-2 therapy (Philip et al., Seminars in Oncology. 24(1 Suppl 4): S32-8, 1997 February) and tumor vaccines targeted against melanoma. Smith et al., Int J Dermatol 1999; 38(7): 490-508. While these strategies are efficacious against some tumors, their potency is limited because they only enhance the already enfeebled ability of tumor cells to present their “foreign” epitopes to CD8+ T-cells, and to generate thereby a tumor-specific cytotoxic T lymphocyte (CTL) response.
Autologous whole tumor cell-based vaccines were first used for immunotherapy of malignant melanoma. Such whole tumor cell-based vaccines are advantageous, because they contain large numbers of antigens, which eliminate the need for targeting the immune response against one antigen at a time. This is important because currently there is little ability to identify specific tumor-associated antigens (TAA) that are useful to induce immune system-mediated tumor regression. Boon et al., Immunol Today 1997; 18:267-268. To date, however, autologous whole tumor cell-based vaccines alone have shown only some isolated or marginal successes. Smith et al., supra. As seen below, the marginal success of whole tumor cell-based vaccines likely results from tumor cell mutations that impair their ability to act as antigen presenting cells (“APCs”).
Evidence from many tumor immunology laboratories demonstrates that tumor cells persist in part because they have selected a mutation which partially or completely destroys their ability to act as APCs in the process of cytotoxic T lymphocyte CTL generation. Stockert et al., J. Exp. Med. 1998; 187: 1349-1354; Sahin et al., Proc. Natl. Acad. Sci. USA 1995; 92:11810-11813; Gabrilovich et al., Nature Med. 1996; 2:1096-1103; Ishida et al., J. Immunol. 1998; 161:4842-4851. These observations spurred development of strategies that attempt to replace the tumor cell as the APC, rather than trying to boost the tumor's enfeebled antigen presenting process. The best candidate for such a replacement is the dendritic cell (“DC”).
DCs are “professional” antigen presenting cells that play a vital role in stimulating immune responses. DCs not only can activate naïve CD4+ T helper cells but also stimulate unprimed CD8+ cytotoxic T lymphocytes. Steinman, R. M. Annu. Rev. Immunol. 1991; 9, 271-296; Macatonia, et al., J. Exp. Med. 1988; 169, 1255-1264; Mehta-Damani et al., J. Immunol. 1994; 153, 996-1003; Porgador et al., J. Exp. Med. 1995; 182, 255-260.
Because of these characteristics, DCs have been widely studied as antigen presenting cells for cancer immunotherapy. DCs can be loaded with tumor antigens by pulsing with whole tumor antigens or tumor antigen peptides. (Young et al., J. Exp. Med. 1996; 183, 7-11; Mayordoma et al., Nat. Med. 1995; 1, 1297-1302; Bakkar et al., Cancer Res. 1995; 55, 5330-5334; Flamand et al., Eur. J. Immunol. 1994; 24, 605-610; Gong et al., Gene Ther. 1997; 4, 1023-1028; Song et al., J. Exp. Med. 1997; 186, 1247-1256; Specht et al. J. Exp. Med. 1997; 186, 1213-1256.)
Peptide- or tumor lysate-pulsed dendritic cells have been used, for example, to vaccinate melanoma patients. (Rosenberg et al., Nature Med 1998; 4: 321-327; Wallack et al., Cancer 1995; 75:34-42; Bystryn, Rec. Results Cancer Res. 1995; 139:337-348; Mitchell et al., Semin. Oncol. 1998; 25: 623-635; Morton et al., Ann. N.Y. Acad. Sci. 1993; 690:120-134; Berd et al., Semin Oncol. 1998; 25:646-653; Berd et al., J. Clin. Oncol. 1997; 15:2359-2370.)
DCs loaded with tumor antigens are able to induce both cellular and humoral, antigen-specific, anti-tumor immune responses. (Shurin, M. R. Cancer Immunol. Immunother. 1996; 43, 158-164). This approach, however, is limited to application against tumors expressing known tumor antigens. See, Haigh et al., Oncology 1999; 13, 1561-1573. It is worthless for those tumors with no identified tumor antigen, like primary tumors from patients, which constitute most real-world situations. Obviously alternative strategies are needed.
An additional problem with antigen pulsing techniques is that the antigen presenting system of an APC works more effectively and efficiently when the protein/antigen is synthesized inside the cell rather than outside the cell, a substantial drawback to using antigen-pulsed cells. In an effort to avoid this problem, a number of laboratories have attempted to use gene therapy to introduce specific tumor antigens into dendritic cells. (Gong et al., 1997, Gene Ther. 4, 1023-28; Song et al., 1997, J. Exp. Med. 186: 1247-56; and Specht et al., 1997, supra.). However, this gene therapy approach is also fraught with many disadvantages including: 1) the limited ability to identify all of the important specific tumor antigens, 2) the limited ability to map the genes of the specific tumor antigens, 3) only one or a small number of the known tumor antigen genes can be introduced into the dendritic cell and 4) the process is time-consuming and cumbersome.
On the other hand, fusions between DCs and tumor cells represent an alternative way to produce effective tumor antigen presenting cells by presenting the immune cells with all possible tumor antigens. (Gong et al., Nat. Med. 1997; 3: 558-561; Wang et al., J. Immunol. 1998; 161, 5516-5524; Lespagnard et al., Int. J. Cancer 1998; 76, 250-258; Rowse et al., Cancer Res. 1998; 58, 315-321). DCs have been fused with tumor cells and the fused cells efficiently presented tumor antigens to the immune cells and stimulated specific anti-tumor immune responses. (Gong et al.; Wang et al.; Lespagnard et al., all supra).
These fusion schemes, however, rely on selectable markers (gene products which render the cell resistant to specific cell toxins or allow them to grow under certain metabolic conditions) in each of the DCs and the tumor cells to isolate the resultant hybrid. The rare cell fusion products are selected by long-term culture in the presence of both cell toxins where only the fusion product, containing both selectable markers, can survive. Since the introduction and selection schemes using markers requires culture and multiple cell division, they cannot be applied to dendritic cells, because DCs are terminally differentiated, non-dividing cells. Thus, it is no surprise that the previous fusion work relied on well-defined tumor cell lines, bearing such a marker, and DC- and tumor-specific conjugated antibodies, which limits the usefulness of this strategy in cancer treatment.
In summary, the previous cancer-based fusion protocols have the following limitations: 1) they require established tumor cell lines which show specific marker(s); 2) they require both DC and tumor cell specific antibodies to select the fused cells; 3) the selection and expansion of the fused cells takes an impractical amount of time.
The area of preventing transplant rejection using hybrids are even less well-developed than cancer. In fact, no report of such has been found.
Typical approaches to preventing transplant rejection utilize non-selective immunosuppressive drugs that suppress the entire immune system. Abbas et al., CELLULAR AND MOLECULAR IMMUNOLOGY, pp. 347-350. Such approaches have the obvious disadvantage of making the patient more susceptible to disorders that otherwise could have been warded off by an intact immune system.
It has been recognized that at least two interactions must take place in order for an antigen presenting cell to activate a T cell. These interactions are between an antigen-loaded major histocompatibility (MHC) antigen and the T cell receptor, and between certain accessory molecules and their cognate receptors on the T cell. The best studied class of these accessory molecules is B7 (B7.1 and B7.2), which interact with CD28 and CTLA4 on T cells. Abbas et al., supra. Thus, disruption of either the MHC or the accessory interaction should result in a non-response useful, for example, in preventing transplant rejection.
In fact, disruption of B7 interaction not only prevents an immune response, it induces permanent tolerance to any antigen presented during the disruption. Wei, et al., 1996, Stem Cells 14: 232-38. Thus, in the context of transplant rejection, blocking B7 should result in tolerance, preventing rejection. The problem with such an approach, and the likely reason that it has not be attempted clinically, is that tolerance would pertain to any antigen presented during treatment, not just to transplant antigens. In other words, if a patient were exposed to a pathogen during the B7 disruption, the patient's immune system would be rendered tolerant to the pathogen, permanently. This would prevent the patient from warding off the pathogen, having perhaps lethal consequences. Clearly, a more specific approach is needed.
A promising approach takes advantage of antigen presentation by cells that lack accessory molecules, like B7. These cells present antigen in the context of MHC, yet, because they lack the accessory interactions required for activation, they induce tolerance specific to the antigen presented. Thus, it is possible to load these cells, which include immature (naïve) B cells, with a specific antigen, and induce antigen-specific anergy. As with the cancer example described above, this antigen-by-antigen approach does not have the general applicability needed for practical clinical use. A methodology is needed which is applicable to any transplant organ, irrespective of the immunogenic antigens the organ displays.
Neuroblastoma is the most common extracranial solid tumor and the most common tumor occurring during infancy. It also affects young children, and is rarely found in children older than 10 years. Neuroblastoma is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts, which are pluripotent sympathetic cells. In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The origin and distribution of these cells during fetal development correlate with the sites of primary disease presentation. The location of tumors appears to vary also with the age of the patient. While most neuroblastomas start in the abdomen, a few neuroblastomas develop in the adrenal glands, abdominal ganglias, chest ganglias, neck, spinal chord or the pelvis. Infants suffer more frequently from thoracic and cervical tumors, whereas older children suffer more frequently from abdominal tumors.
Age, stage, and some molecular defects in the tumor cells are the prognostic factors used for risk assessment and treatment strategy. The differences in outcome between patients with neuroblastoma are striking. Infants younger than 1 year have a good prognosis, even in the presence of metastatic disease, whereas older patients with metastatic disease fare poorly, even when treated with aggressive therapy. Unfortunately, approximately 70-80% of patients older than 1 year suffer from metastatic disease, usually to lymph nodes, liver, bone, and bone marrow. Fewer than half of these patients are cured, even with the use of high-dose therapy followed by autologous bone marrow or stem cell rescue.
Treatments for neuroblastoma include surgery, chemotherapy, and/or radiation therapy. Surgery is often used to try to remove as much as possible of the tumor in combination with adjuvant chemotherapy. Chemotherapy becomes the main treatment when the cancer has spread too far to be completely removed by surgery. Most common drugs used in chemotherapy include cyclophosphamide or ifosfamide, cisplatin or carboplatin, vincristine, doxorubicin, etoposide, teniposide and topotecan. A typical combination of drugs commonly used consists of cyclophosphamide, doxorubicin, and vincristine and is alternated with cisplatin plus etoposide. Common side effects include nausea, vomiting, hair loss, mouth sores, depression of the immune system, and bone marrow suppression. In addition, ifosfamide and cyclophosphamide may produce bladder inflammation and blood in the urine, and damage to the kidneys with subsequent loss of salt and minerals in the urine. Cisplatin may produce hearing loss or deafness, kidney damage, and severe and delayed nausea. Doxorubicin (Adriamycin) may cause heart damage if too much of the drug is given and can cause skin damage if the drug should leak out of the vein during administration.
Accordingly, there is a need in the art for improved treatment options for cancer patients, including neuroblastoma patients, and the present invention satisfies that need. There is also a need in the art for rapid methods for inducing and suppressing specific immune responses to whole cells and specific reagents for accomplishing these methods.